Explanation To investigate pathways dependent on the PAR-degrading activity of ARH3 under oxidative stress induced by H2O2 exposure, we generated mouse embryonic fibroblasts (MEFs) from wild-type (WT) and ARH3-/- littermates. In WT MEFs, although ARH3 has a mitochondrial targeting sequence at its N-terminus, most of the ARH3 was found in cytoplasm (65%), followed by mitochondria (25%) and nucleus (10%). Exposure to H2O2 (100-1000 &#956;M) for 24 h induced greater cytotoxicity with ARH3-/- than WT MEFs and over-expression of ARH3 in ARH3-/- MEFs reduced sensitivity to H2O2-induced cell death. All findings were consistent with a physiological role for ARH3 in resistance to cell death under oxidative stress induced by H2O2 exposure. Since oxidative stress induced by H2O2 exposure can result in cell death by necrosis and/or apoptosis, we assessed the mode of H2O2-induced cell death in ARH3-/- MEFs. After 3 or 6-h exposure to 300 &#956;M H2O2, ARH3-/- MEFs, but not WT MEFs and ARH3-/- MEFs expressing ARH3, exhibited nuclear shrinkage, chromatin condensation, and exposure of phosphatidylserine on the cell surface, which are common characteristics of apoptotic cells. Incubation with the caspase inhibitor zVAD-fmk for 1 h before H2O2 exposure did not improve viability of WT or ARH3-/- MEFs. Furthermore, H2O2 exposure did not enhance PARP1 cleavage, a hallmark of an early stage of caspase-dependent apoptosis. These results indicate that H2O2-induced death of ARH3-/- MEFs resulted from caspase-independent apoptosis. Oxidative stress induced by H2O2 exposure causes DNA damage, followed by PAR synthesis. We investigated whether ARH3 deficiency altered cellular PAR metabolism after H2O2 exposure. Before H2O2 exposure, reaction of MEFs with anti-PAR antibodies appeared faint, and confined to the cytoplasm. Exposure to 300 &#956;M H2O2 increased PAR content primarily in nuclei of WT and ARH3-/- MEFs, as soon as 10 min after addition of H2O2. PAR content of nuclei reached a peak at 20 min and gradually decreased to basal levels within 1 h. At all times after H2O2 exposure, however, nuclear PAR content was much greater in ARH3-/- than WT MEFs. After 20 min, cytoplasmic PAR levels of ARH3-/- MEFs appeared to increase somewhat, concomitant with decreasing nuclear PAR, whereas PAR distribution in WT MEFs did not change. Over-expression of ARH3 protein in ARH3-/- MEFs suppressed the early increase in nuclear PAR as well as its slower accumulation in cytoplasm, effects not seen in ARH3-/- MEFs transfected with empty vector. Western blot analysis confirmed that H2O2 exposure induced greater and more prolonged elevation of PAR in ARH3-/- than WT MEFs or ARH3-/- MEFs over-expressing ARH3. All data were consistent with the notion that ARH3 participates in nuclear and cytoplasmic PAR degradation following H2O2 exposure, although it does not appear to regulate basal PAR levels. As our results strongly suggested PAR participation in the induction of death of ARH3-/- MEFs following H2O2 exposure, we explored the role of PARP in cell viability during oxidative stress. Pharmacological inhibition of PARP by PJ34 significantly increased the number of viable ARH3-/-, but not WT MEFs. In contrast to the effects of PARP inhibition by PJ34, neither PARP2-specific (UPF1035) nor tankyrase-specific (XAV939) inhibitors altered the sensitivity to H2O2-induced cell death. Next, we assessed the effects of PARP1 depletion with shRNA on ARH3-/- MEFs. Depletion of PARP1 protein by shRNA reduced ARH3-/- MEF sensitivity to H2O2-induced cell death, with significantly less nuclear shrinkage and chromatin condensation than seen in ARH3-/- MEFs transformed with control shRNA. Using immunocytochemistry to evaluate localization of intracellular PAR, we found that depletion of PARP1 protein resulted in reduction of nuclear PAR content and decreased accumulation of cytoplasmic PAR. Western blot analysis also showed reduced and more transient PAR levels in ARH3-/- MEFs expressing PARP1 shRNA. Taken together, all results are consistent with the conclusion that PARP1, but neither two other PARP proteins nor SIRT1, is responsible for PAR production and for initiation of cell death in ARH3-/- MEFs after H2O2 exposure. ARH3 appears to confer protection against PARP1-mediated cell death, by preventing accumulation in the nucleus of excess PAR and its translocation to cytoplasm and mitochondria. After PARP1 activation, excessive PAR production results in caspase-independent cell death, which is mediated by release of AIF from mitochondria. AIF was restricted to mitochondria of WT and ARH3-/- MEFs under resting conditions, and, after exposure to 300 &#956;M H2O2 for 3 or 6 h, was accumulated in nuclei of ARH3-/-, but not WT MEFs. In addition, ARH3 expression or depletion of PARP1 protein in ARH3-/- MEFs prevented AIF accumulation in nuclei. Longer exposure to higher concentrations of H2O2 (600 &#956;M, 6 h) increased nuclear content of AIF in both WT and ARH3-/- MEFs expressing ARH3 protein, suggesting that AIF accumulation depends on the intensity of the stimulus and lack of ARH3 protein or activity enhances the magnitude of the effect. It appears that cell death resulting from ARH3 deficiency is associated with a caspase-independent pathway involving PARP1 activation and release of AIF from mitochondria and translocation to nuclei. To explore the relationship between ARH3 and PARG, we generated ARH3-/- MEFs stably expressing PARG shRNA. Western blot analysis revealed that PARG depletion in ARH3-/- MEFs led to a slight increase in basal PAR content as well as sustained PAR elevation following H2O2 exposure. ARH3-/- MEFs expressing PARG shRNA exhibited reduced sensitivity to H2O2 exposure and less nuclear shrinkage in response to H2O2 than did ARH3-/- MEFs expressing control shRNA. Furthermore, ARH3-/- MEFs expressing PARG shRNA failed to AIF accumulation in the nucleus. These results indicate that, unlike ARH3 deficiency, depletion of PARG by shRNA protected against H2O2-induced cell death by preventing AIF release from mitochondria. PAR translocation to the cytoplasm and mitochondria was essential to trigger AIF release from mitochondria. Using immunocytochemistry, we found that the accumulation of nuclear PAR content in ARH3-/- MEFs expressing PARG shRNA was greater and more prolonged than those of ARH3-/- MEFs expressing control shRNA. ARH3-/- MEFs expressing PARG shRNA, however, failed to show slower accumulation of PAR in the cytoplasm; thus, PARG appeared to regulate PAR translocation from the nucleus to cytoplasm. In addition, 2 h-exposure to H2O2 failed to induce PAR accumulation in mitochondria of ARH3-/- MEFs expressing PARG shRNA. Since the majority of PAR is attached to PARP1 itself and PARG catalyzes hydrolysis of more protein-bound than protein-free PAR, we assumed a role of PARG in the release of protein-free PAR from poly-ADP-ribosylated PARP1. Poly-ADP-ribosylation of PARP1 in both WT and ARH3-/- MEFs was seen as soon as 20 min of H2O2 exposure. ARH3-/- MEFs expressing PARG shRNA maintained the levels for more than 1 h, whereas poly-ADP-ribosylation of PARP1 in MEFs expressing control shRNA was significantly elevated only at 20 min. Our findings indicate that PARG regulates PAR release from poly-ADP-ribosylated PARP, and thereby its levels in nuclei and cytoplasm. In summary, ARH3 regulates nuclear and cytoplasmic PAR degradation and confers a protective effect against H2O2-induced cell death. ARH3 regulates the levels of PAR following its release from poly-ADP-ribosylated acceptor proteins by action of PARG. ARH3 is responsible for modulating cytoplasmic PAR content and localization, thereby regulates AIF release from mitochondria. These studies are the first evidence for a physiological function of ARH3.